CN102947452A - Methods and compositions using listeria for adjuvant treatment of cancer - Google Patents

Abstract

Provided herein are prime-boost regimens and materials used therein. The prime-boost regimens enhance the immune response to a target antigen. The vaccines used for boost are comprised of recombinant attenuated metabolically active Listeria that encodes an expressible antigen that is cross-reactive with the target antigen. In some examples, the immune response is a cellular immune response.

Description

Use the method and composition of the listeria of assisting a ruler in governing a country treatment that is used for cancer

The present invention requires the right of priority of the U.S. Provisional Patent Application 61/347,447 of submission on May 23rd, 2010, comprises that its full content of all forms, accompanying drawing and claim is incorporated into hereby.

Statement about federal government's sponsored research

United States Government has full-payout license licensed licenser licence of the present invention and has under the suitable clause that provides according to National Cancer Institute (the National Cancer Institute) number of authorizing NHI 1K23CA104160-01 and require other people right of prior licensing from the patentee in the situation of restriction.

Background of invention

It is believed that the strong cellular immunization of setting up specific pathogen is required.Verified, using the repetitive administration (homology reinforcement) of identical vaccine is effective for strengthening humoral response.Yet, present generation with suitable inflammation signal because before the immunity of carrier is tended to damage strong antigen, so the method is to strengthening cellular immunization relative nullity.A kind of method of evading this problem is that continuous administration is used the not vaccine of synantigen dependency system (heterology reinforcement).The method is called " just exempting from-strengthen (prime-boosting) ".

Below be that heterology is just exempted from-example of strengthened scheme.Those that relate to that DNA just exempts from comprise: DNA just exempts from: and the DNA for virus antigen just exempts from/and bacterium strengthens (listeria) (Boyer etc. (2005) Virology 333:88-101); DNA just exempts from/bacteria carrier (bacillus) reinforcement, directed toward bacteria antigen (Ferraz etc. (2004) Infection Immunity72:6945-6950); DNA just exempts from/the virus vector reinforcement, for tumour antigen (Goldberg etc. (2005) Clin.Cancer Res.11:8114-8121; Smith etc. (2005) Int.J.Cancer113:259-266); DNA for virus antigen just exempts from/virus reinforcement (Toussaint etc. (2005) Vaccine 23:5073-5081; Cebere etc. (2006) Vaccine 24:417-425; Coupar etc. (2006) Vaccine 24:1378-1388); DNA for virus antigen just exempts from/albumen reinforcement (Cristillo etc. (2006) Virology 346:151-168; Rasmussen etc. (2006) Vaccine 24:2324-2332); DNA just exempts from/the virus reinforcement, for parasitic antigen (Gilbert etc. (2006) Vaccine 24:4554-4561; Webster etc. (2005) Proc.Natl.Acad.Sci.USA 102:4836-4841); The albumen that DNA just exempts from/assists a ruler in governing a country is strengthened, for tumour antigen (Prud'homme (2005) J.Gene Med.7:3-17); DNA just exempts from/virus reinforcement and albumen reinforcement, for virus antigen (Stambas etc. (2005) Vaccine23:2454-2464); And DNA just exempts from (nano particle)/albumen and strengthens, for virus antigen (Castaldello etc. (2006) Vaccine 24:5655-5669).

Following heterology just exempts from-and the strengthened scheme utilization do not relate to the first composition of exempting from of DNA: and dendritic cell (DC) just exempts from/bacterium (listeria) is strengthened and DC just exempts from/and virus strengthens directed toward bacteria antigen (Badovinac etc. (2005) Nat.Med.11:748-756); Bacteria carrier is just exempted from (salmonella)/albumen and is strengthened directed toward bacteria antigen (Vindurampulle etc. (2004) Vaccine 22:3744-3750; Lasaro etc. (2005) Vaccine 23:2430-2438); The albumen of assisting a ruler in governing a country is just exempted from/the DNA reinforcement, for virus antigen (Sugauchi etc. (2006) J.Infect.Dis.193:563-572; Pal etc. (2006) Virology 348:341-353); Albumen is just exempted from/bacteria carrier (salmonella) reinforcement, for virus antigen (Liu etc. (2006) Vaccine24:5852-5861); Albumen is just exempted from/the virus vector reinforcement, for virus antigen (Peacock etc. (2004) J.Virol.78:13163-13172); Heterology virus is just exempted from/the virus reinforcement, uses the different virus carrier, for virus antigen or tumour antigen (Ranasinghe etc. (2006) Vaccine24:5881-5895; Kaufman etc. (2004) J.Clin.Oncol.22:2122-2132; Grosenbach etc. (2001) Cancer Res.61:4497-4505).Use the heterology of lipid vesicle just to exempt from/strengthen, directed toward bacteria antigen (Luijkx etc. (2006) Vaccine 24:1569-1577).

To be used for immune reagent be listeria and be especially listeria monocytogenes (Listeria monocytogenes).Listeria monocytogenes have to liver and spleen and to a certain degree to such as the natural tropism of its hetero-organization of small intestine (referring to, for example, Dussurget etc. (2004) Ann.Rev.Microbiol.58:587-610; Gouin etc. (2005) Curr.Opin.Microbiol.8:35-45; Cossart (2002) Int.J.Med.Microbiol.291:401-409; Vazquez-Boland etc. (2001) Clin.Microbiol.Rev.14:584-640; Schluter etc. (1999) Immunobiol.201:188-195).Be positioned at enteron aisle for bacterium, then by the listeria albumen such as ActA and internalin A mediate passage to blood flow (referring to, for example, Manohar etc. (2001) Infection Immunity69:3542-3549; Lecuit etc. (2004) Proc.Natl.Acad.Sci.USA101:6152-6157; Lecuit and Cossart (2002) Trends Mol.Med.8:537-542).In case bacterium enters host cell, the life cycle of listeria monocytogenes comprises from phagolysosome escapes to cytosol.This life cycle in contrast to Mycobacterium (Mycobacterium), its still in phagolysosome (referring to, for example, Clemens etc. (2002) Infection Immunity 70:5800-5807; Schluter etc. (1998) Infect.Immunity 66:5930-5938; Gutierrez etc. (2004) Cell 119:753-766).Listeria monocytogenes from phagolysosome escape by such as pig Listeria monocytogenes element (lysteriolysin) (LLO), the listeria protein mediated (referring to (2002) J.Cell Biol.158:409-414 such as Portnoy) of PI-PLC and PC-PLC.

Contrast immunotherapy method discussed above, wherein immunotherapy is as primary treatment; Adjuvant therapy referred to before main therapy (" neoadjuvant ") or afterwards (" assisting a ruler in governing a country ") use less important treatment, for example operation or radiation, wherein main therapy is intended to remove or destroy main tumour.For example, after operation, can provide adjuvant therapy, wherein remove detectable disease, but owing to hide the statistics risk that also there is recurrence in disease.For the eurypalynous cancer of being permitted that comprises colorectal carcinoma, lung cancer, carcinoma of the pancreas, mammary cancer, prostate cancer and some gynecological cancers, provide subsystem therapy and radiation-therapy after the operation.

Brief summary of the invention

The invention provides the adjuvant therapy scheme that adopts the listeria bacterium to be used for the secondary treatment of cancer.Using once to the experimenter or repeatedly before or after the elementary treatment, using the listeria vaccine of the significant quantity of expressing cancer associated antigens.The present invention includes the test kit based on the vaccine of listeria of containing that is packaged in the suitable container, and can comprise specification sheets.

In first aspect, the present invention relates to suffer from cancered mammiferous method of assisting a ruler in governing a country treatment, the method was included in before or after the main therapy of described administration and treats to remove or kill the cancer cell of expressing cancer antigen as assisting a ruler in governing a country to the composition of described administration significant quantity, described composition comprise attenuation, the metabolic activity listeria, described attenuation, metabolic activity listeria encode effable, the immunocompetence part of described cancer antigen.

As mentioned above, composition of the present invention can be provided as neoadjuvant; Yet in preferred embodiments, after main therapy, use composition of the present invention.In each embodiment, described main therapy comprises the operation of removing described mammiferous described cancer cell; Kill the radiation-therapy in cancer cell described in the described Mammals; Perhaps described operation and described radiation-therapy.

Because listeria can be causal organism, and particularly in immunoincompetent situation, preferred described step of applying comprise with multiple doses use described attenuation, the metabolic activity listeria, described attenuation, metabolic activity listeria encode effable, the immunocompetence part of described cancer antigen.But " attenuation " refers to modify by this bacterium to alleviate or to eliminate its pathogenicity bo still keep method as the ability of the prevention of target disease or treatment.Can obtain the bacterium attenuation by different mechanisms.A kind of is to introduce sudden change to one or more pathways metabolisms, and its function is crucial for existence in the bacterial body and growth to cause a disease.In the situation of listeria, in certain embodiments, make mutant bacteria to alleviate or to prevent the ability of Intracellular growth and diffusion.Preferred listeria can comprise the sudden change that makes the ActA inactivation; Make the sudden change of InlB inactivation; Perhaps both.Most preferably, adopt in the present invention its natural A ctA of disappearance and inlB gene (Δ actA Δ inlB) attenuation, the metabolic activity listeria.In certain embodiments, present method adopts and is killed but the listeria (" KBMA ") of tool metabolic activity.

One or more nucleic acid recombinant expressed of any type of the various cancer antigens of coding is provided by composition of the present invention.In certain embodiments, cancer antigen all or a part be mesothelin.Other suitable antigens are described in detail as follows, and can be depending on type of cancer to be treated and pass through cancer antigen to be expressed.In each embodiment, cancer to be treated is selected from carcinoma of the pancreas, nonsmall-cell lung cancer, ovarian cancer and mesothelioma.This inventory is not to be intended to restriction.

Except being preferably operation or the main therapy of radiation as described, composition of the present invention can be sent as just exempting from-part of strengthened scheme.For example, using between the listeria composition of the present invention, expressed in advance the cell from the human cell line of target cancer antigen to administration, the reorganized modification of wherein said cell is to generate and the secretion rHuGM-CSF, and wherein modified described cell by radiation in case cell fission (namely

Vaccine, Cell Genesys, Inc.).

Should be understood that the present invention when it is used, be not limited to structure detail and set forth in the following description or accompanying drawing in the layout of illustrated component.The present invention can have embodiment (except those embodiments of describing) and can and carry out with the whole bag of tricks enforcement.And, should be understood that wording and the term that this paper adopts and make a summary to be used for the purpose of describing and should not to be considered as restriction.

In itself, it will be understood by those skilled in the art that can easily utilize the disclosure based on concept carry out other structures of multiple purpose of the present invention, the basis of method and system as design.Therefore, importantly, as long as they do not depart from the spirit and scope of the present invention, claim just should be deemed to be included in these equivalent constructions.

The accompanying drawing summary

Figure 1A is the figure to the OVA specificity cellular immunity response of target antigen (OVA) that show to use due to the listeria monocytogenes of antigen expressed and first the exempting from of vaccinia virus-strengthened scheme.The OVA specificity that is presented in the C57BL/6 mouse of using OVA257-264 peptide (SIINFEKL (SEQ IDNO:1)) inoculation in the ICS mensuration is replied.

Figure 1B is the figure that shows by the people's mesothelin specificity cellular immunity response due to the listeria monocytogenes of use expression target antigen (people's mesothelin) and first the exempting from of adenovirus-strengthened scheme.

Fig. 2 is the figure of people's mesothelin specificity cellular immunity response in the Balb/c mouse that shows by due to first the exempting from of using listeria monocytogenes, adenovirus and vaccinia virus-strengthened scheme.Also diagram is just exempted from-strengthened scheme.

Fig. 3 A shows by the adenovirus of using the increase level just to exempt from the figure (using mesothelin peptide pond (peptide pool) to stimulate splenocyte) of people's mesothelin specificity cellular immunity response in C57BL/6 (HLA-A2 transgenosis) mouse due to first the exempting from strengthened with the listeria monocytogenes of constant level-strengthened scheme.

Fig. 3 B shows by the adenovirus of using the increase level and just exempts from people's mesothelin specificity cellular immunity response in the Balb/c mouse (stimulating splenocyte with mesothelin peptide pond) due to first the exempting from strengthened with the listeria monocytogenes of constant level-strengthened scheme.Also diagram is just exempted from-strengthened scheme.

Fig. 4 A is presented at the left side of figure, its show by have be pre-existing in to the people's mesothelin specificity cellular immunity response due to adenovirus is just exempted from the mouse of the immunity of adenovirus.Also in Fig. 4 A, show such as the adenovirus specificity of type of service I Hex3 epi-position (adenovirus specificity epitope) mensuration and reply.Also diagram is just exempted from-strengthened scheme.

Fig. 4 B be presented at have be pre-existing in to the people's mesothelin specificity cellular immunity response due to just being exempted to strengthen with listeria monocytogenes by adenovirus in the mouse of the immunity of adenovirus and in and the figure of the titre of adenovirus specific antibody.Also diagram is just exempted from-strengthened scheme.

Fig. 5 shows by use

Listeria monocytogenes first of (CELL GENESYS, INC.) or coding AH1 exempted from and uses

The figure of the AH1 specificity cellular immunity response due to the reinforcement of the listeria monocytogenes of (CELLGENESYS, INC.) or coding AH1.

Fig. 6 A be show by use through the adenovirus of the dendritic cell of mesothelin peptide 131-139 pulse or encoding human mesothelin first exempt from and use due to the reinforcement of listeria monocytogenes of encoding human mesothelin at CD8 ⁺Mesothelin in the T cell _131-139The percentile figure that specificity is replied.Just exempt to occur in the 0th day; Strengthen occurring in the 8th day; And at the 13rd day results splenocyte.

Fig. 6 B shows the figure that exempts from and use the absolute number of mesothelin l31-139 specific C D8+T cell in each spleen due to the reinforcement of listeria monocytogenes of encoding human mesothelin through the adenovirus of the dendritic cell of mesothelin peptide 131-139 pulse or encoding human mesothelin first by using.Just exempt to occur in the 0th day; Strengthen occurring in the 8th day; And at the 13rd day results splenocyte.

Fig. 6 C shows by using through the dendritic cell of mesothelin peptide 131-139 pulse first to exempt from and use the percentile figure that mesothelin 131-139 specificity is replied in the CD8+T cell due to the reinforcement of following coding mesothelin reagent: listeria monocytogenes, adenovirus or vaccinia virus.

Fig. 7 A is the figure that is presented at splenocyte small mouse mesothelin specific immune response, wherein is evaluated at the immunne response of each peptide in the mouse mesothelin peptide library.The Balb/c mouse is accepted the first reinforcement of exempting from and using the listeria monocytogenes of encoding murine mesothelin of the naked DNA carrier of use encoding murine mesothelin.

Fig. 7 B is the figure that is presented at splenocyte small mouse mesothelin specific immune response, wherein is evaluated at the immunne response of each peptide in the mouse mesothelin peptide library.The Balb/c mouse is accepted the first reinforcement of exempting from and using the listeria monocytogenes of encoding murine mesothelin of the adenovirus of use encoding murine mesothelin.Also diagram is just exempted from-strengthened scheme.

Fig. 7 C is that the shadow tone of the result's that shows that enzyme linked immunological spotting method (elispot) is measured hole photograph is reproduced (hali-tone reproduction), and the splenocyte that wherein obtains from mouse (its result of study is presented at Fig. 7 B) is exposed to mesothelin peptide numbering 278 (SEQ ID NO:2), 279 (SEQ ID NO:3) or 280 (SEQ ID NO:4).

Fig. 8 be show by the KBMA listeria monocytogenes that uses coding OVA as just exempt from and KBMA listeria monocytogenes, vaccinia virus or " works " listeria monocytogenes as the figure of the OVA specificity cellular immunity response due to first the exempting from of reinforcement-strengthened scheme.All " reinforcement " vector encoded OVA.

Fig. 9 A is presented at that AH-and AH 1/A5-specific T-cells reply in the Mice Inoculated.

Fig. 9 B is presented in the Mice Inoculated mesothelin specific T-cells and replys.

Fig. 9 C is presented at the Survival data that the CT26 tumour excites the mouse that (tumor challenge) inoculate afterwards.

Detailed Description Of The Invention

The present invention relates to be used to causing the materials and methods as the immunne response of the target antigen of non-listeria antigen.Target antigen is preferably the target antigen with disease-related, so that provide therapeutic action to the immunne response of target antigen.The invention provides the vaccine group of assisting a ruler in governing a country treatment of cancer, wherein the vaccine based on listeria is the metabolic activity listeria, the encode immunocompetence part of the target antigen of expressing among its host who is applied of this metabolic activity listeria.

The present invention's part can strengthen the discovery that a large amount of primary carcinoma are treated based on the auxiliary use of using vaccine, and this vaccine is comprised of the listeria monocytogenes of the attenuation of the work of the relevant antigen (for example mesothelin) of encoding cancer.

Except as otherwise noted, otherwise the routine techniques comprise molecular biology (comprising recombinant technology), immunology, cytobiology, biological chemistry and medicine rules is adopted in enforcement of the present invention.The complete explanation of these technology in the literature, for example, Molecular Cloning:A Laboratory Manual, second edition, (Sambrook etc.); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (editor such as F.M.Ausubel); Current Protocols in Immunology (John Wiley ﹠amp; Sons, Inc., N.Y.); Handbook of Pharmaceutical Excipients (editor such as Rowe); Vaccine (Plotkin and Orenstein, 2003); And Vaccine Protocols (Methods in Molecular Medicine) (Robinsin, Cranage and Hudson, 2003).

Definition

The abbreviation that is used for being illustrated in the gene sudden change or suddenlys change in comprising the bacterium of gene is as follows.For example, abbreviation " listeria Δ actA " refers to disappearance part or all actA genes.Triangle symbol (Δ) expression disappearance.Comprise subscript negative sign (listeria actA ^-) abbreviation refer to this actA transgenation, for example disappearance, point mutation or phase shift mutation, but be not limited to the sudden change of these types.Index can be abridged, for example " 3e7 " refers to 3x10 ⁷

As this paper that comprises claims uses, and except text offers some clarification in addition, comprises plural indicator such as the singulative of the vocabulary of " one ", " a kind of ", " described ".The bibliography that this paper quotes by reference integral body is incorporated into, and the degree of incorporating into is as pointing out that clearly and individually each independent sequence open, that register by the GenBank accession number, patent application, patent, sequence table, Nucleotide in sequence table or oligopeptides or peptide sequence and picture and photo in described open and patent documentation incorporate into by reference.

When being applied to people, Mammals, mammalian subject, animal, beasts experimenter, placebo subjects, research experimenter, experimental subjects, cell, tissue, organ or biofluid, " using " refers to that (but being not limited to) makes the exogenous lipid of contact, reagent, placebo, small molecules, pharmaceutical agent, therapeutical agent, diagnostic reagent or the compositions such as experimenter, cell, tissue, organ or biofluid." use " can represent for example to treat, prevention, pharmacokinetics, research, placebo and experimental technique.Cell " using " contained make the reagent exposing cell; And reagent contacting with fluid, wherein this fluid contact cell." use " the external and stripped method that also contains by reagent, bonding composition or the cell by another cell.Such as comparing of using or do not use with the use placebo, can be by the survival time (for example, to life-threatening proliferative illness) that for example increases; Reduce tumor size; Reduce the tumour number; Reduce the metabolism of specific tissue; Reduce the metabolism of specific tissue; The titre of reduction infective agent etc. is assessed the result who uses." treatment " contained using of expection effect." treatment " comprises preventative (preventing disease) and treats and use.

" antigen presenting cell " is for the immune cell of antigen-presenting to the T cell.APC comprise dendritic cell, monocyte, scavenger cell, marginarium Kupffer cell, microglia, Langerhans cell, T cell and B cell (referring to, for example, Rodriguez-Pinto and Moreno (2005) Eur.J.Immunol.35:1097-1105).Dendritic cell appears in two pedigrees at least.The first pedigree comprises pre-DCl, marrow DC1 and ripe DC1.The second pedigree comprises CD34 ⁺⁺CD45RA ^-Early stage multi-functional T progenitor cell, CD34 ⁺⁺CD45RA ⁺Cell, CD34 ⁺⁺CD45RA ⁺⁺CD4 ⁺IL-3Ralpha ⁺⁺Pro-DC2 cell, CD4+CD11c ^-Plasmacytoid pre-DC2 cell, lymph people DC2 Plasmacytoid source property DC2 and ripe DC2 (referring to, for example, Gilliet and Liu (2002) J.Exp.Med.195:695-704; Bauer etc. (2001) J.Immunol.166:5000-5007; Arpinati etc. (2000) Blood95:2484-2490; Kadowaki etc. (2001) J.Exp.Med.194:863-869; Liu (2002) Human Immunology 63:1067-1071; McKenna etc. (2005) J.Virol.79:17-27; O'Neill etc. (2004) Blood 104:2235-2246; Rossi and Young (2005) J.Immunol.175:1373-1381; Banchereau and Palucka (2005) Nat.Rev.Immunol.5:296-306).

" attenuation " and " attenuation " contained modified to reduce gene in bacterium to host's toxicity, virus, parasite, infection biological, tumour cell, the infection biological etc.The host can be people or animal host or organ, tissue or cell.Provide non-limitative example, can make the bacterium attenuation with the combination of reduction with host cell; Diffuse to another host cell to reduce from a kind of host cell; To reduce the extracellular growth in the host cell; Perhaps to be reduced in the Intracellular growth of host cell.By measuring such as toxicity index, LD ₅₀, clearance rate or coefficient of competition can be assessed attenuation (referring to, for example, Auerbuch etc. (2001) Infect.Immunity 69:5953-5957) from organ.Usually, attenuation causes LD ₅₀Increase at least 25%; More generally at least 50%; The most common at least 100% (2 times); At least 5 times of routines; More conventional at least 10 times; The most conventional at least 50 times; Often at least 100 times; More often at least 500 times; And the most at least 1000 times; Generally at least 5000 times; More general at least 10,000 times; And the most general at least 50,000 times; And the most common at least 100,000 times.

" gene of attenuation " comprises that mediation is to host's toxicity, pathology or virulence; In the host, grow; The gene of perhaps surviving in the host, wherein gene suddenlys change in the mode that alleviates, reduces or eliminate toxicity, pathology or virulence." gene of sudden change " is included in the control region of gene, the coding region of gene, the non-coding region of gene or disappearance, point mutation, insertion mutation and the phase shift mutation in its any combination.

" carninomatosis disease " and " cancer illness " includes but not limited to cancer; Tumour; The transfer of tumour, vasculogenesis; And such as illness before the dysplastic cancer.Mammalian subject with cancer, tumour, the front illness of cancer, the front illness of cancer or cancer illness etc. contains the mammalian subject that not only comprises cancer, the front illness of cancer or tumour, and contain and remove tumour, obviously eliminate the mammalian subject of cancer (for example, by chemotherapy or operation or separately for experimenter's immunity system).

As " significant quantity " in treatment, used include but not limited to improve, reverse, mitigation or the symptom of preventive medicine illness or illness or the amount of sign.Unless have in addition clearly or otherwise and illustrate that " significant quantity " is not limited to enough improve the minimum of illness; Best or the maximum amount of improving that perhaps causes illness." significant quantity " is the amount that causes immunne response in the Mammals in the text of using of just exempting from and/or strengthening.

" extracellular fluid " contains fluid, lymph liquid, bile, sweat, fecal matter and the urine of for example serum, blood plasma, blood, interstitial fluid, cerebrospinal fluid, secretion." extracellular fluid " can comprise colloid or suspended matter, for example whole blood.

" growth " of listeria bacterium is the term in listeria field, and it is encompassed in the Intracellular growth of the listeria bacterium that grows in the host cell such as mammalian cell.Although can measure by optical microscopy, fluorescent microscopy or colony-forming unit (CFU) assay method the Intracellular growth (" growth ") of listeria bacterium, any technical limitation that growth is not measured.Amount, listeria nucleotide sequence or the specific biochemical parameter of listeria bacterium lipid such as listeria antigen can be used for the assessment growth.The gene of mediating growth is the gene of growth in the specificity mediated cell.The gene of growth includes but not limited to that wherein gene inactivation reduces Intracellular growth speed but cannot not reduce the gene of extracellular growth velocity (for example, the growth in the meat soup) with detecting in the specificity mediated cell; Perhaps wherein the degree of the speed of gene inactivation reduction Intracellular growth is compared the larger gene of degree that it reduces the speed of extracellular growth.For non-limitative example is provided, the gene of growth is that wherein its disappearance reduces Intracellular growth to less than 50%, less than 40%, less than 30%, less than 20% or less than the gene of 10% the Intracellular growth that is shown by the wild-type listeria in the mediated cell.For further non-limitative example is provided, the disappearance that the gene of growth is contained wherein it in the mediated cell not only reduces Intracellular growth to the Intracellular growth less than 50% wild-type listeria, and reduces the gene that the extracellular grows to the extracellular growth that about 95%, 90%, 85% or 80% use wild-type listeria detects.In the text, term " about " refers to ± 5%.

" kill but the tool metabolic activity " and (KBMA) contain any genomic bacterium that contains modification, for example wherein genomic modification is enough to prevent bacterium colony to form, but wherein genomic modification is not enough to basically stop or destroy metabolism.The KBMA bacterium can not form such as detectable bacterium colony on agar or in the host cell body.For non-limitative example is provided, can use the linking agent such as psoralene to come the modifying factor group.For non-limitative example is provided, the KBMA bacterium of all not destroyed in metabolism is that wherein genome only contains crosslinked a kind of in the intergenic region that does not have regulation and control, coding, structure or biological function.This in contrast to " work " bacterium, and it can form bacterium colony, although the bacterium that lives in some embodiments can be for attenuation.

" sample " refers to the sample from people, animal, placebo or study sample, for example, and cell, tissue, organ, fluid, gas, aerosol, slurry, colloid or the material that condenses." sample " also refers to for example comprise the cell of fluid or tissue sample or the cell that separates from fluid or tissue sample." sample " also can represent cell, tissue, organ or the fluid of fresh sampling from the human or animal; Perhaps treated or the cell, tissue, organ or the fluid that store.

Bacterium " diffusion " contains " cell is to cellular invasion ", and it is that part is transferred to the second host cell by the bacterium from the first host cell that vesica mediates.The function relevant with diffusion includes but not limited to: for example, and the formation of Actin muscle tail; The formation that the pseudopodium sample prolongs; And the formation of two film vacuoles.

" just exempt from dosage " or " significant quantity " of " just exempting from consumption " refers to cause the amount of the target antigen that can measure immunne response as comparing with immunne response in not having the mammalian subject of administration of antigens in mammalian subject.

As used herein " vaccine " refers to the composition that formed or be comprised of the nucleic acid of encoding heterologous albumen by heterology albumen, so that when to the administration vaccine, heterology albumen expresses to be used for causing the purpose of immunne response in Mammals.Immunne response can for body fluid, cell-mediated or both.

As used herein " first immune seedling (priming vaccine) " refers to comprise to cause the vaccine of the reagent that the significant quantity of the immunne response of target antigen is used to experimenter or host.First immune seedling codified antigen, and the also various immunostimulatory cell factors of codified such as GM-CSF, it is used for replenishing and activation antigen is delivery cell.

As used herein " strengthen vaccine (boosting vaccine) " refers to comprise the vaccine of reagent of antigen that coding has the immunocompetence part of target antigen, and this reagent can comprise target antigen, its fragment and/or contain the fusion polypeptide of at least immunocompetence part of the target antigen that is bonded on common non-existent zone in the target antigen." significant quantity " of " booster dose " or " reinforcement consumption " is in case refer to the administration of having used before the target antigen of just exempting from dosage then cause amount to the antigen of the immunne response of target antigen.

As used herein " vehicle group " or " vaccine group " comprise just to be exempted from carrier or first immune seedling and strengthens carrier or the reinforcement vaccine, and wherein each own coding is shared immunologic determinants, cross reaction immunologic determinants, shared antigen, immune protein or one of peptide or its fragment.

" antigen " refers to contain to stimulate such as the host immune system of the immune system molecule with one or more epi-positions of producing body fluid and/or cell antigen specificity and replying (linear, conformation or both) or immunologic determinants.The commutative use of this term and term " immunogen ".Antigen can be the fragment of whole protein, truncated protein or albumen or peptide.Antigen can be naturally occurring, the genetically engineered variant of albumen; Perhaps can be the optimizing codon that is used for particularly expressing mammalian subject or host.Usually, the B cell epitope comprises about 5 amino acid at least, but also can be less to 3-4 amino acid.T cell epitope such as the CTL epi-position comprises at least about 7-9 amino acid, and helper T cell epitope comprises at least about 12-20 amino acid.Usually, epi-position comprises about 7 and 15 amino acid, for example, and 9,10,12 or 15 amino acid.Two subunit antigen of term " antigen " expression (that is, from complete organism, separating and discrete antigen and the antigen relevant with natural antigen).But the antibody such as the synthetic peptide mimic epitopes of antiidiotypic antibody or its fragment and analogue antigen or antigenic determinant also is encompassed under the definition of as used herein antigen.For the present invention, antigen can be from any one of multiple known pathogenic virus, bacterium, parasite and fungi.They also can be from cancer.And, for the present invention, " antigen " refer to as long as albumen keep the ability that causes immunne response then comprise to naturally occurring sequence usually occurring in nature conservative such as disappearance, add and the as herein defined albumen of the modification that replaces.These modifications can for what have a mind to, for example be passed through site-directed mutagenesis; Perhaps can be for chance, for example sudden change of the host by producing antigen.Antigen of the present invention also can for by the optimized codon of means known in the art to improve their expression or the immunogenicity in the host.As used herein, " cross reaction " immunologic determinants refers to cause that for example the CRD of HIV is the antigen that can cause the immunne response of two or more among all members of HIV antigen in the differentiation branch to relevant but the not determinant of the immunne response of same antigen determinant, epi-position or antigen.

To antigen or be present in the development of body fluid and/or the cellullar immunologic response of the antigen in the vehicle group in mammalian subject to antigen, carrier or vaccine or " immunity is replied " or " immunne response " that comprise the composition of antigen." cellullar immunologic response " is by the T-lymphoblast and/or includes but not limited to that other white cells of NK cell and scavenger cell mediate.T lymphoblast of the present invention comprises the T cell of express alpha β φt cell receptor subunit or expresses T cell and the effector of γ δ acceptor or suppress sub-T cell." T lymphoblast " or " T cell " is that non-antibody generates lymphoblast, and it consists of the part of immune cell-mediated arm.The T cell comes from immature lymphoblast, and this maturation lymphoblast migrates to thymus gland by marrow, wherein the ripening process of they experience under the orientation of thymine.Based on the ability of their identification and binding specificity antigen, mature T cells becomes and has immunological competence.When antigen during in conjunction with lymphoblastic surface receptor, trigger the activation of immunocompetent T cell.Known, in order to produce t cell response, must be in cell synthetic antigen or antigen must be introduced in the cell, by the proteasome mixture it is treated to little peptide subsequently, and is displaced to endoplasmic reticulum/Golgi mixture Secretory Pathway to be used for last and the association of ajor histocompatibility mixture (MHC) Grade I albumen.The functioning cell immunity comprises antigen-specific cytotoxic t lymphocytes (CTL).When albumen from the mankind in the time, as used herein T cells with antigenic specificity, CTL or cytotoxic T cell refer to having specific cell with albumen by ajor histocompatibility mixture (MHC) or human leucocyte antigen (HLA) (HLA) the coding peptide antigen that exists that associates.CTL of the present invention comprises the CTL of activation, and it is triggered by specific antigens in the environment of MHC; But and be called owing to the memory CTL of the T cell that is exposed to again the reactivation that becomes among antigen and the cross reaction CTL or recover CTL.CTL of the present invention comprises CD4+ and CD8+T cell.The Peptide-specific CTL of activation of the present invention promotes destruction and/or the cracking of the experimenter's that CTL infects its special pathogenic agent or cancer cell cell by the following: the especially secretion of chemokine and cytokine (including but not limited to macrophage inflammatory protein 1a (MIP-1a), MIP-1B and RANTES); And the secretion that suppresses the soluble factor of morbid state.Cellular immunization of the present invention refers to reply by the antigen-specific of the T auxiliary subunit generation of T cell.Helper cell be used for to help stimulatory function, and concentrate on the surface that is presented at them with the activity of the nonspecific effect cell of the T cell of the peptide of MHC molecular association.Cellullar immunologic response is the phalangeal cell factor, chemokine and by the T cell that activates and/or comprise the generation of other these molecules that generated by cell-derived those other white cells of CD4 and CD8 T cell and NK also.What cause cellullar immunologic response just exempts from dosage or booster dose or comprises composition or the vaccine of just exempting from dosage or booster dose to can be used for the sensitization mammalian subject by the antigen of presenting at cell surface place and MHC molecular association.Cell-mediated immunne response is oriented in or near the cell of surface antigen-presenting at them.In addition, antigen specific T-lymphoblast is so that can further protect the host of immunity.By measuring the ability that the immunity of specific antigen irritation cell mediation is replied such as lymphadenosis (lymphoblast activation) assay method, CTL cytotoxic cell assay method or by the specific many measure method known in the art of the T-lymphoblast of antigen among the experimenter who is determined at sensitization.These assay methods are well known in the art.Referring to, for example, Erickson etc., J.Immunol. (1993) 151:4189-4199; Doe etc.; Eur.J.Immunol. (1994) 24:2369-2376.The method of measuring cell-mediated immunne response comprises by the T-cell colony measures the cell within a cell factor or cytokine secretion; Perhaps by measure epitope specificity T cell (for example, passing through Tetramer technology) (by McMichael, A.J., and O'Callaghan, C.A., J.Exp.Med.187 (9) 1367-1371,1998; Mcheyzer-Williams, M.G. etc., Immunol.Rev.150:5-21,1996; Lalvani, A. etc., J.Exp.Med.186:859-865,1997 summaries).As used herein immunity is replied or immunne response contains the generation of the generation that stimulates CTL and/or helper cell and/or antibody-mediated immunne response or the immunity of activation is replied or immunne response.

As used herein " immunity is replied " or " immunne response " contain at least a or multiple of following effect: by B Hemapoiesis antibody; And/or the directed antigen of suppressor T cell and/or specificity or be present in the activation of the T cell of the antigen in destination carrier, composition or the vaccine.In some embodiments, " immunity is replied " or " immunne response " contains the inactivation of suppressor T cell.As used herein, " booster immunization of enhancing is replied " refer to by booster dose cause as with the using of the vaccine of replying the larger measurable immunne response of comparing that causes by the single administration of just exempting from dosage.

So-called " pharmaceutically acceptable " or " acceptable on the pharmacology " refers to not to be biologically to expect or the other material of expectation.

As used herein, term " test kit " is although refer to pack and/or component mark and the common use of unnecessary while.Test kit can comprise just immune seedling and reinforcement vaccine in independent container.Test kit also can comprise first immune seedling and/or strengthen the component of vaccine in independent container.Test kit can comprise for merging component and being suitable for to the component of the immunogenic composition of Mammals use with preparation.

" therapeutic action " is one or more symptoms of slowing down with the disease-related of vaccine to be administered." prophylactic effect " is the inhibition to one or more symptoms of the disease relevant with vaccine to be administered.

" mammalian subject " as used herein or " host " refer to any member of subphylum Chordata, include but not limited to people and other primatess that comprise such as the non-human primates of chimpanzee and other apes and monkey class; Domestic animal, for example ox, sheep, pig, goat and horse; Domestic Mammals, for example dog and cat; The laboratory animal that comprises rodent, for example mouse, rat and cavy.Term does not represent the specific age.Therefore, be intended to comprise adult and newborn individuality.

" radiation-therapy " as used herein or " radiotherapy " refer to as the medical usage of the ionizing rays of the part of cancer therapy with the control malignant cell.Radiotherapy can be used for healing property, complementary or retentivity treatment.Radiotherapeutic adequate types comprises conventional external beam radiotherapy; Stereotactic radiation-therapy (for example, Axesse, ejected wave cutter (Cyberknife), gamma knife, Novalis, Primatom, Synergy, X-Knife, TomoTherapy or Trilogy); The radiation-therapy of intensity modulated; Part Ther (for example, proton therapy); Brachytherapy (brachytherapy); Send radio isotope etc.This inventory is not to be intended to restriction.

Cause the method to the immunne response of target antigen

The present invention is contained for the method that causes immunne response Mammals.Target antigen can be those relevant with morbid state, for example, and as having those that are identified at cancer cells or pathogenic agent.After the primary carcinoma treatment, use the vaccine of the metabolic activity listeria of the immunocompetence attenuation partly that comprises coding and express target antigen.

1. target antigen

During the example of the target antigen that can use in treatment plan of the present invention is listed in the table below.Target antigen also can be immunocompetence fragment or the fusion polypeptide partly that is included in the antigen of listing in the table.

Table 1. antigen

2. the reagent of just exempting from-using in the reinforcement approach

Just exempting from-the reinforcement approach in, adjuvant therapy can comprise the first immune seedling to the administration effective dose.Initial vaccine does not preferably contain the metabolic activity listeria of the target antigen of encoding.These vaccines can contain himself target antigen, for example, have or do not have the albumen, tumor cell lysate of adjuvant, through the tumour cell of radiation, use the antigen presenting cell of target cell (for example dendritic cell) pulse; Perhaps it can contain the reagent that target antigen is provided.Provide the suitable reagent of target antigen to comprise recombinant vectors, for example, bacterium, virus and naked DNA.Prepare recombinant vectors with standard technique known in the art, and this recombinant vectors contains the suitable controlling elements that is operatively connected to the nucleotide sequence of coding target antigen.Referring to, for example, Plotkin etc. (editor) (2003) Vaccines, the 4th edition, W.B.Saunders, Co., Phila., PA.; Sikora etc. (editor) (1996) Tumor Immunology Cambridge University Press, Cambridge, UK; Hackett and Harn (editor) Vaccine Adjuvants, Humana Press, Totowa, NJ; Isaacson (editor) (1992) Recombinant DNA Vaccines, Marcel Dekker, NY, NY; Morse etc. (editor) (2004) Handbook of Cancer Vaccines, Humana Press, Totowa, NJ); Liao etc. (2005) Cancer Res.65:9089-9098; Dean (2005) Expert Opin.Drug Deliv.2:227-236; Arlen etc. (2003) Expert Rev.Vaccines 2:483-493; (2003) Vaccine21:1317-1326 such as Dela Cruz; Johansen etc. (2000) Eur.J.Pharm.Biopharm.50:413-417; Excler (1998) Vaccine 16:1439-1443; Disis etc. (1996) J.Immunol.156:3151-3158).Peptide vaccine be described in (referring to, for example, McCabe etc. (1995) Cancer Res.55:1741-1747; Minev etc. (1994) Cancer Res.54:4155-4161; Among Snyder etc. (2004) the J.Virology 78:7052-7060.

The virogeny carrier comprises virus, modified virus and virus particle (referring to, for example, table 2).The virogeny carrier directly can be used to mammalian subject; Perhaps it can be exsomatized and introduce in the antigen presenting cell (APC), wherein then use APC to the experimenter.

Virus vector can be based on for example, and Togaviruses comprises alphavirus and arboviruses; Alphavirus, for example sindbis alphavirus (Sindbis virus), Syndebis bacterial strain SAAR86, Semliki Forest virus (Semliki Forest virus) (SFV), Venezuelan equine encephalitis (VEE), eastern equine encephalitis (EEE), western equine encephalitis, ross river virus, sagiyama virus (Sagiyami virus), Ao Niyongniyong virus (O'Nyong-nyong virus), Highland J virus; Arboviruses, for example yellow fever virus, yellow jack bacterial strain 17D, Japanese encephalitis, SLE, tick encephalitis, dengue virus, west Nile virus, Kunjin virus (hypotype of west Nile virus); Arterivirus is equine arteritis virus for example; And for example Ankara bovine vaccine (MVA) of rubella virus, simplexvirus, modification of rubella virus genus; The fowlpox virus carrier; The fowl pox carrier; Vaccinia virus vector; Influenza vectors; Adenovirus carrier; The human papilloma virus poisonous carrier; Bovine papilloma virus carrier etc.Virus vector can be based on orthopoxvirus, for example variola virus (smallpox), vaccinia virus (vaccine of smallpox), Ankara (MVA) or copenhagen strain, ostrich flower, monkeypox or cowpox.Virus vector can be based on sparrow pox virus, for example fowlpox virus or canary pox virus.

Can obtain the adenovirus carrier virus vector relevant with gland (AAV), wherein adenovirus carrier comprises adenoviral serotype 5 (adeno5; Ad5), adeno6, adeno11 and adeno35.Can obtain at least 51 kinds of adenovirus hominis serotypes, it is classified into six subgroups (subgroup A, B, C, D, E and F).The immunne response adenovirus protein that for example can be used for assessing " blank " adenovirus carrier comprises hexon, for example six adjacent body 3 albumen, scleroproein and penton base albumen, and described human immune to adenovirus protein (referring to, for example, (2002) J.Virol.76:12775-12782 such as Wu; Mascola (2006) Nature 441:161-162; Roberts etc. (2006) Nature 441:239-243).

Table 2. virogeny vaccine carrier

Antigen presenting cell (APC) carrier such as dendritic cell (DC) carrier comprises the cell that carries through antigen; Cell through the Tumor lysate carrying; The perhaps cell of the composition transfection through comprising nucleic acid, its amplifying nucleic acid can be for example plasmid, mRNA or virus.Also can use DC/ tumour fusion bacterin.Referring to, for example, (2004) the Clin.Cancer Res.10:5381-5390 such as Di Nicola; Cerundolo etc. (2004) Nature Immunol.5:7-10; Parmiani etc. (2002) J.Natl.Cancer Inst.94:805-818; Kao etc. (2005) Immunol.Lett.101:154-159; Geiger etc. (2005) J.Transl.Med.3:29; Osada etc. (2005) the Cancer Immunol.Immunother.11 month 5, electronic publication before the 1-10[printing]; Malowany etc. (2005) Mol.Ther.13:766-775; Morse and Lyerly (2002) World J.Surg.26:819-825; Gabrilovich (2002) Curr.Opin.Mol.Ther.4:454-458; Morse etc. (2003) Clin.Breat Cancer 3 supplement 4:S164-S172; Morse etc. (2002) Cancer Chemother.Biol.Response Modif.20:385-390; Arlen etc. (2003) Expert Rev.Vaccines 2:483-493; Morse and Lyerly (1998) Expert Opin.Investig.Drugs 7:1617-1627; Hirschowitz etc. (2004) J.Clin.Oncol.22:2808-2815; Vasir etc. (2005) Br.J.Haematol.129:687-700; Koido etc. (2005) Gynecol.Oncol.99:462-471.

Can obtain as vaccine such as the tumour cell (Arlen etc. (2005) Semin.Oncol.32:549-555) from body and allogeneic tumour cell.Vaccine also can comprise the tumour cell of modification, for example tumor cell lysate or through the tumour cell of radiation.By incorporating coding into such as cytokine (GM-CSF, IL-12, IL-15 etc.); The NKG2D lipid; CD40L; CD80; The nucleic acid of the molecule of CD86 etc. also can modify tumour cell (referring to, for example, Dranoff (2002) Immunol.Rev.188:147-154; Jain etc. (2003) Ann.Surg.Oncol.10:810-820; Borrello and Pardoll (2002) Cytokine Growth Factor Rev.13:185-193; Chen etc. (2005) Cancer Immunol.Immunother.27:1-11; Kjaergaard etc. (2005) J.Neurosurg.103:156-164; Tai etc. (2004) J.Biomed.Sci.11:228-238; Schwaab etc. (2004) J.Urol.171:1036-1042; Friese etc. (2003) Cancer Res.63:8996-9006; Briones etc. (2002) Cancer Res.62:3195-3199; Vieweg and Dannull (2003) Urol.Clin.North Am.30:633-643; Mincheff etc. (2001) Crit.Rev.Oncol.Hematol.39:125-132).

Vaccine can comprise naked DNA carrier and naked RNA carrier.Can by particle gun, electroporation, bacterial ghost, microsphere, particulate, liposome, polycation nano particle etc. use these vaccines of containing nucleic acid (referring to, for example, Donnelly etc. (1997) Ann.Rev.Immunol.15:617-648; Mincheff etc. (2001) Crit.Rev.Oncol.Hematol.39:125-132; Song etc. (2005) J.Virol.79:9854-9861; Estcourt etc. (2004) Immunol.Rev.199:144-155).

Can obtain to use by particle gun, intradermal, intramuscular and electroporation method reagent and the method for naked nucleic acid.Nucleic acid vaccine can comprise lock nucleic acid (LNA), and wherein LNA be so that can make funtion part be attached to plasmid DNA, and wherein funtion part can for adjuvant (referring to, for example, Fensterle etc. (1999) J.Immunol.163:4510-4518; Strugnell etc. (1997) Immunol.Cell Biol.75:364-369; Hertoughs etc. (2003) Nucleic Acids Res.31:5817-5830; Trimble etc. (2003) Vaccine 21:4036-4042; Nishitani etc. (2000) Mol.Urol.4:47-50; Tuting (1999) Curr.Opin.Mol.Ther.1:216-225).Nucleic acid vaccine can be united the reagent that promotes that immature dendritic cell moves towards vaccine; And promote ripe DC to migrate to just to exempt from the reagent of draining lymph node that can appearance place to use, wherein these reagent comprise MIP-1 α and Flt3L (referring to, for example, Kutzler and Weiner (2004) J.Clin.Invest.114:1241-1244; Sumida etc. (2004) J.Clin.Invest.114:1334-1342).

Bacteria carrier comprises, for example, and salmonella, Shigella, Yersinia, lactobacillus genus, streptococcus, bacille Calmette-Guerin vaccine, Bacillus anthracis and intestinal bacteria.Can be with the bacterium through engineering approaches to contain the coding recombinant antigen; Heterogenetic antigen; The nucleic acid of the antigen of perhaps being derived by tumour, cancer cell or infective agent.And, can modify bacterium with attenuation.In another aspect, non-listeria bacterial vaccine can lack any coding recombinant antigen nucleic acid (referring to, for example, Xu etc. (2003) Vaccine 21:644-648; Pasetti etc. (2003) J.Virol.77:5209-5219; Loessner and Weiss (2004) Expert Opin.Biol.Ther.4:157-168; Grangette etc. (2002) Vaccine 20:3304-3309; Byrd etc. (2002) Vaccine 20:2197-2205; Edelman etc. (1999) Vaccine 17:904-914; Domenech etc. (2005) Microbes and Infection 7:860-866).

Can by the bacterium preparation of living kill but tool metabolic activity (" KBMA ") bacterium and particularly KBMA listeria by (for example using the DNA linking agent, psoralene) processes and/or by such as the recombinational repair gene (for example making, recA) or UV-light injury repairing gene (for example, uvrA, uvrB, uvrAB, uvrC, uvrD, phrA, at least a gene inactivation that mediated dna phrB) is repaired (referring to, for example, the U.S. Patent Publication No. 2004/0228877 and 2004/0197343 of Dubensky etc., it incorporates this paper into way of reference integral body separately).

Class in the KBMA listeria is the listeria uvrAB through through engineering approaches of expressing heterologous antigen, wherein use nucleic acid linking agent, psoralene compound, mustard compound, 4'-(4-amino-2-oxa-) butyl-4,5', two (2-chloroethyl) amino of 8-trimethylpsoralen or Beta-alanine, N-(acridine-9-yl), 2-[] ethyl ester processes the bacterium of through engineering approaches.Also referring to, for example, the U.S. publication application number US 2004/0197343 of Dubensky etc., MODIFIED FREE-LIVING MICROBES, VACCINE COMPOSITIONS AND METHODS OF USE THEREOF; Brockstedt etc. (2005) Nature Med.11:853-860).

In some embodiments, first immune seedling comprise be selected from poxvirus (VV) carrier, dendritic cell (DC) carrier, adenovirus carrier, naked DNA carrier and

The reagent of (CELL GENESYS, INC.).

3. the listeria of in adjuvant therapy, using

In certain embodiments, the present invention includes and use the listeria bacterium, wherein listeria is through attenuation.Attenuation can be by due to one or more genes of the viral factor of sudden change coding, for example actA, internalization protein B (inlB), p60 (autolysin), pig Listeria monocytogenes element O (LLO; The hly gene), phosphatidylcholine Phospholipase C (PC-PLC), phosphatidylinositol-specific phospholipase C (PI-PLC; The plcA gene), Thioctic Acid protein ligase and be disclosed in the ENGINEERED LISTERIA AND METHODS OF USE THEREOF that transfers Cerus Corporation, gene in the U.S. serial 11/395,197 (submission on March 30th, 2006).Method of the present invention includes but not limited to the bacterial strain that uses one or more listeria kinds and this paper to identify.For example, the present invention is contained and is used the listeria bacterium be or derived by listeria monocytogenes.Also can use such as through through engineering approaches to express one or more pig Listeria monocytogenes element O (hly gene; Other kinds of the listeria of harmless listeria spp LLO); PlcA; PlcB; Perhaps such as viral gene or mediation enter the gene in the host cell other genes (referring to, for example, Johnson etc. (2004) Appl.Environ.Microbiol.70:4256-4266; Slaghuis etc. (2004) J.Infect.Dis.189:393-401; Milohanic etc. (2003) Mol.Microbiol.47:1613-1625).Can be as preparing the bacterial strain of the attenuation that is suitable for the listeria of in reinforcement vaccine of the present invention, using as described in PCT/US2004/003429 and the PCT/US2004/044080.All above-mentioned applications are all incorporated this paper into way of reference integral body.

The non-limitative example of the listeria of attenuation is described in the following patent disclosure (it incorporates this paper into way of reference integral body separately): U.S. Patent Publication No. 2004/0228877; U.S. Patent Publication No. 2004/0197343; With U.S. Patent Publication No. 2005/0249748.Also provide non-limitative example in the Application No. 11/395,197 of submitting to such as on March 30th, 2006, it incorporates this paper into way of reference integral body.

4. vaccine composition

Except mentioned reagent, vaccine composition of the present invention can further comprise various vehicle, adjuvant, carrier, subsidiary material, modulator etc.The optional carrier that exists is the molecule that self can not induce the deleterious effect of the individuality that receives composition.Suitable carrier is generally macromole large, slow metabolism, for example protein, polysaccharide, poly(lactic acid), polyglycolic acid, polyamino acid, amino acid copolymer, lipid aggregate (for example oil droplet or liposome) and nonactive virus particle.The example of specific support comprise derived by poly methyl methacrylate polymer those and by PLA and be called the particulate that the PLG of PLG is derived, referring to, for example, Jeffery etc., Pharm.Res. (1993) 10:362-368; McGee J P etc., J Microencapsul.14 (2): 197-210,1997; O'Hagan D T etc., Vaccine 11 (2): 149-54,1993.These carriers be known to a person of ordinary skill in the art those.In addition, these carriers can be used as immunostimulant (" adjuvant ").And, can make antigen with from the bacterial toxoid of diphtheria, tetanus, cholera etc. and the anatoxic bacterial toxoid conjugation of being derived by intestinal bacteria.These adjuvants include but not limited to: (1) aluminium salt (alum), such as aluminium hydroxide, aluminum phosphate, Tai-Ace S 150 etc.; (2) the O/w emulsion formulation (has or does not have other specific immunity stimulants, for example Muramyl dipeptide (see lower) or bacterial cell wall fraction), for example (a) uses such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.) microfluidizer is mixed with submicron 5% squalene, 0.5% tween 80 and the 0.5% sorbester p37 MF59 (international publication number WO 90/14837) of (although do not need, the optional MTP-PE (seeing lower) that contains various amounts) that contains; (b) miniflow changes into microemulsion or vortex to generate the more SAF that contains 10% squalane, 0.4% tween 80,5% pluronic segmented copolymer L121 and MDP of macroparticle size emulsion; And (c) contain 2% squalene, 0.2% tween 80 and one or more are selected from monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton, the Ribi.TM. that is preferably the bacterial cell wall fraction of MPL+CWS (Detoxu) assists a ruler in governing a country system (RAS), (Ribi Immunochem, Hamilton, MT); (3) can use such as the saponin adjuvant of Stimulon.TM. (Cambridge Bioscience, Worcester, Mass.) or generate thus particle such as ISCOM (immunostimulating complex); (4) Freund＇sadjuvantcomplete (Complete Freunds Adjuvant) (CFA) and Fei Shi Freunds incomplete adjuvant (IFA); (5) cytokine is such as interleukin-class (IL-1, IL-2 etc.), macrophage colony stimulating factor (M-CSF), tumour necrosis factor (TNF), β chemokine (MIP, 1-α, 1-β chemokine etc.); (6) such as the bacterium ADP-glycosylation toxin of Toxins,exo-, cholera (CT), Toxins, pertussis (PT) or HLT (LT) through separating virus mutants, particularly LT-K63 (wherein 63 place's Methionins are replaced by wild-type amino acid in the position); LT-R72 (wherein 72 place's arginine are replaced by wild-type amino acid in the position); CT-S109 (wherein 109 place's Serines are replaced by wild-type amino acid in the position); And PT-K9/G129 (wherein 9 place's Methionins are replaced by wild-type amino acid and 129 place's glycine are substituted in the position in the position) (referring to, for example, international publication number WO93/13202 and WO92/19265); And (7) are used as other materials of immunostimulant with the effect of enhancing composition.

5. the preparation of vaccine composition

Prepare vaccine by method known to those skilled in the art.Usually, prepare one or more mentioned reagent (being used for just exempting from or strengthening vaccine) by reagent and the pharmaceutically acceptable vehicle that mixes aequum.Pharmaceutically acceptable vehicle includes but not limited to sterile distilled water, salt solution, phosphoric acid buffer, based on amino acid whose damping fluid or bicarbonate buffer.

6. vaccine administration

Those skilled in the art can be determined at the first of significant quantity to be supplied in the vaccine of one or more dosage and exempt from carrier or strengthen carrier.This tittle falls in the scope that can measure by routine test.

Can measure just immune seedling and strengthen vaccine by any one or combination in the following path.On the one hand, use just immune seedling and reinforcement vaccine by same paths.In another aspect, use just immune seedling and reinforcement vaccine by different paths.Term " different path " includes but not limited to different positions on health, such as in (lymphoglandula), intravenously in oral, non-oral, the intestines, in the parenteral, rectum, node, artery, subcutaneous, intramuscular, the tumour, around the tumour, in the tumour, infusion, mucous membrane, nose, in myelencephalon space or cerebrospinal fluid etc.; And by different mode, for example oral, intravenously and intramuscular.

Can one dosage give significant quantity just exempt from or strengthen vaccine, but be not limited to a dosage.Therefore, this use can for twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, ten once, ten secondaries, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times or using of vaccine more frequently.When vaccine administration is once above, can one minute, two minutes, 3,4,5,6,7,8,9,10 or timed interval of more minutes; Separate with one hour, two hours, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours etc. interval and to use.Hour situation in, term " about " refers to about arbitrary time span in 30 minutes.Also can separate and use by the timed interval of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days and combination.The present invention is not limited to dosing interval and evenly separates in time, also contains the not administration of same intervals, for example by to use the Initially immunity time table that forms in 1 day, 4 days, 7 days and 25 days, only is used for providing non-limitative example.

Below when determining the relative timing of first immune seedling and reinforcement vaccine, should considering.It is found that, but the expansion of using stimulator antigen specific immunity cell of the nucleic acid of antigen or coding for antigens causes occurring peak value, be subsequently the antigen specific immune cell number reduction (referring to, for example, (2002) Nature Immunol.3:619-626 such as Badovinac).Before peaking, with peak value simultaneously or after peak value, can use the startup of booster shot.

When the population expansion (increasing on the number) of antigen specific immune cell at least 20% of the maximum number of the final antigen specific immune cell that obtains; To at least 30%; To at least 40%; To at least 50%; To at least 60%; To at least 70%; To at least 80%; To at least 90%; To at least 95%; During to the maximum number of the antigen specific immune cell of at least 99% final acquisition, can start using of booster shot.Can obtain just to exempt from-strengthen the other timetable of vaccine, for example, be reduced to when the population of antigen-specific sexual cell under the maximum number of 90% antigen-specific sexual cell; Under 80%; Under 70%; Under 60%; Under 50%; Under 40%; Under 30%; Under 20%; Under 10%; Under 5%; Under 1.0%; Under 0.5%; Under 0.1%; Under 0.05%; Can start booster shot in the time of perhaps under the maximum number of 0.01% antigen specific immune cell.The antigen-specific sexual cell can be identified as that carrier specificity antigen (to the specificity of blank carrier) is had specificity; Perhaps to having specificity by the heterogenetic antigen that is included in the expression of nucleic acid in the carrier.

In other respects, start just exempt from inoculation after about 5 days; Start just exempt from inoculation after about 10 days; After starting using of just exempting to inoculate about 15 days; About 20 days; About 25 days; About 30 days; About 35 days; About 40 days; About 45 days; About 50 days; About 55 days; About 60 days; About 65 days; About 70 days; About 75 days; About 80 days; About 6 months; And can start using of booster shot about 1 year the time.

just exempt from the inoculation after 5-10 days; just exempt from the inoculation after 10-15 days; just exempt from the inoculation after 15-20 days; just exempt from the inoculation after 20-25 days; just exempt from the inoculation after 25-30 days; just exempt from the inoculation after 30-40 days; just exempt from the inoculation after 40-50 days; just exempt from the inoculation after 50-60 days; After just exempting from inoculation, 60-70 days etc. can use booster shot.

Those skilled in the art can be determined to start and just exempt to inoculate and start time period between the booster shot.For example, it can be based on the algorithm to the physiological parameter sensitivity of mensuration after the immunity at the beginning of occuring.

Usefulness by mode, vaccine delivery, the theme demand of employing and depend on that operator's judgement determines dosage and scheme at least in part.

For example, can dosage or dosage use listeria in the vaccine that uses in the present invention, wherein each dosage comprises 10 ⁷-10 ⁸Individual listeria/70kg body weight; 2x10 ⁷-2x10 ⁸Individual listeria/70kg body weight; 5x10 ⁷-5x10 ⁸Individual listeria/70kg body weight; 10 ⁸-10 ⁹Individual listeria/70kg body weight; 2.0x10 ⁸-2.0x10 ⁹Individual listeria/70kg body weight; 5.0x10 ⁸-5.0x10 ⁹Individual listeria/70kg body weight; 10 ⁹-10 ¹⁰Individual listeria/70kg; 2x10 ⁹-2x10 ¹⁰Individual listeria/70kg; 5x10 ⁹-5x10 ¹⁰Individual listeria/70kg; 10 ¹¹-10 ¹²Individual listeria/70kg; 2x10 ¹¹-2x10 ¹²Individual listeria/70kg; 5x10 ¹¹-5x10 ¹²Individual listeria/70kg; 10 ¹²-10 ¹³Individual listeria/70kg; 2x10 ¹²-2x10 ¹³Individual listeria/70kg; 5x10 ¹²-5x10 ¹³The weight in wet bases such as individual listeria/70kg.Based on per 1.7 per square meter of surface area meters, or based on 1.5kg liver restatement, also provide above each dosage.Note, when using listeria of the present invention, heavily about 1.5 grams of mouse liver.About 1.5 kilograms of people's liver weight.

In some embodiments of the present invention, the listeria of booster dose strengthens at least 2 times; About 3 to 5 times or 5 times to 10 times; The perhaps first dosage immunne response of exempting from of 10 times to 100 times or larger multiple.In some embodiments of the present invention, just exempt from dosage and booster dose and have synergistic effect to immunne response.In some embodiments of the present invention, the immunne response of enhancing comprises the T-cell response; And in some embodiments, the T-cell response is CD8 ⁺The T-cell response.In some embodiments of the present invention, just exempt from dosage and booster dose and destroy Mammals for the tolerance status of target antigen.The example of all these embodiments below is provided.

7. measure the method for immunne response

Various external and in vivoassay methods for measuring immunne response (comprise and measure body fluid and cellullar immunologic response) are known in this area, and it includes but not limited to the standard immunoassay assay method, for example RIA, ELISA assay method; Cell inner dyeing; Comprise for example T raji cell assay Raji of Lymphoid tissue propagation (lymphoblast activation) assay method; CTL cytotoxic cell assay method; Perhaps by measuring the antigen in the sensitization experimenter had specific T-lymphoblast.These assay methods are well known to a person skilled in the art.Referring to, for example, Erickson etc., J.Immunol. (1993) 151:4189-4199; Doe etc., Eur.J.Immunol. (1994) 24:2369-2376.The current method of measuring cell-mediated immunne response comprises the measurement of the cell within a cell factor or the cytokine by the secretion of T cell population; The perhaps measurement (for example, passing through Tetramer technology) by epitope specificity T cell (by McMichael, A.J. and O'Callaghan, C.A., J.Exp.Med.187 (9) 1367-1371,1998; Mcheyzer-Williams, M.G. etc., Immunol.Rev.150:5-21,1996; Lalvani, A. etc., J.Exp.Med.186:859-865,1997 summaries).In schematic embodiment disclosed herein, enzyme linked immunological spot (ELISPOT) assay method is for detection of secreting Interferon, rabbit with analyzing

Independent cell.The enzyme linked immunological spotting method Assay method and reagent can be by BDBiosciences 2350 Qume Drive San Jose, CA, and 95131 obtain.The ELISPOT assay method can detect the cell of the founder cell factor and by adopting high affinity capture and detecting antibody and enzymatic amplification obtains its specificity and susceptibility from the T cell population first contact test and memory that activates.Be provided at J.Immunol.Methods.2001,254 (1-2): in 59 about the other information of using the ELISPOT assay method.Animal model such as non-human primates is known in the art.For example, mouse is the acceptable model for human immune.NK cells in mice is that NK cells of human beings is to the acceptable model of replying of tumour to replying of tumour.In addition, mouse T cell is human T-cell's model; Mouse dendritic cell (DC) is the model of people DC; Mouse NKT cell is the model of people NKT cell; Congenital the replying of mouse is the congenital acceptable model of replying of people etc.Model research openly is used for for example CD8 ⁺The T cell; The maincenter memory T cell; And Effector memory T cell (referring to, for example, Walzer etc. (2002) J.Immunol.168:2704-2711); Two kinds of hypotypes of NK cell (referring to, for example, Chakir etc. (2000) J.Immunol.165:4985-4993; Smith etc. (2000) J.Exp.Med.191:1341-1354; Ehrlich etc. (2005) J.Immunol.174:1922-1931; Peritt etc. (1998) J.Immunol.161:5821-5824); The NKT cell (referring to, for example, Couedel etc. (1998) Eur.J.Immunol.28:4391-4397; Sakamoto etc. (1999) J.Allergy Clin.Immunol.103:S445-S451; Saikh etc. (2003) J.Infect.Dis.188:1562-1570; Emoto etc. (1997) Infection Immunity 65:5003-5009; Taniguchi etc. (2003) Annu.Rev.Immunol.21:483-513; Sidobre etc. (2004) Proc.Natl.Acad.Sci.101:12254-12259); Monocyte/macrophage (Sunderkotter etc. (2004) J.Immunol.172:4410-4417); Two pedigrees of DC (Boonstra etc. (2003) J.Exp.Med.197:101-109; Donnenberg etc. (2001) Transplantation 72:1946-1951; Becker (2003) Virus Genes 26:119-130; Carine etc. (2003) J.Immunol.171:6466-6477; Penna etc. (2002) J.Immunol.69:6673-6676; Alferink etc. (2003) J.Exp.Med.197:585-599).Congenital the replying of mouse that comprises Toll sample acceptor (TLR) is the model of people's innate immune responses, as be disclosed in (referring to, for example, Janssens and Beyaert (2003) Clinical Microb.Revs.16:637-646) in.The mouse neutrophilic leukocyte be the human neutrophil acceptable model (referring to, for example, Kobayashi etc. (2003) Proc.Natl.Acad.Sci.USA 100:10948-10953; Torres etc. (2004) 72:2131-2139; Sibelius etc. (1999) Infection Immunity 67:1125-1130; Tvinnereim etc. (2004) J.Immunol.173:1994-2002).But mouse to the immunne response of listeria be the people to the Acceptance Model of replying of listeria (referring to, for example, Kolb-Maurer etc. (2000) Infection Immunity 68:3680-3688; Brzoza etc. (2004) J.Immunol.173:2641-2651).

8. just exempt from-use of strengthened scheme

Can treat and/or suppress cancer and infection by using the immune reagent of regulation and control.Be encompassed in interior first the exempting from of the present invention-Jia strong method and cause the immunne response of rise, and comprise the tolerance that destroys autoantigen.Therefore, expect that these first exempting from-Jia strong methods can be used for suppressing growth and/or improvement one or more symptoms relevant with cancer of cancer.Also expection, just exempt from-the Jia strong method can be used for preventing and/or treating the disease that is caused by pathogenic agent.

Except above, these schemes can be used for determining whether Mammals replys treatment.For example, when using first exempting to specific antigens-Jia strong method, obtain the significance immunne response and unsuccessfully show after strengthening, Mammals is not replied target antigen and selectable mode that should continual cure.When the genetic background of cancer or pathogenic agent so that target antigen lacks or when not modifying with the mode of target antigen cross reaction, can be this example.

Embodiment

Should be understood that the present invention is not limited to specific examples disclosed herein, therefore can change.Also should be understood that because scope of the present invention illustrate by claims, so these examples be not be intended to restricted.

The general information of the method for using in an embodiment

Assess immunne response to vaccine by the results splenocyte, this splenocyte provides the immune cell that comprises T cell and dendritic cell (DC).By hatching splenocyte with one or more peptides and measuring immunologic cellular activity and measure antigen-specific immune response, wherein measure activity by cell inner dyeing (ICS) and enzyme linked immunological spot assay method.In some assay methods, only add single peptide, wherein peptide only contains an epi-position of tumour antigen.In other assay methods, add the whole library of the peptide of the whole length that comprise antigen.

Comprising of ICS assay method splenocyte, and with processing in conjunction with the antibody that is gathered in the cytokine in the immunocyte, wherein antibody allows fluorescent mark.The brefeldin blocks protein is transported, and causes the gathering in the immunocyte inner cell factor.

Elispot (enzyme linked immunological spot) assay method is responsive for the albumen of secretion, wherein immunocyte is being placed hole for some time begin secretory protein.Capture antibodies is bonded to the hole, the cytokine of its immobilization secretion.After Secretory cycle, cell is removed, and use and detect antibody to detect fixing cytokine.The not same district of capture antibodies and detection antibodies cytokine.The method details (referring to, for example, the disclosed U.S. Patent Application Publication No. 2005/0249748 in 10 days November in 2005 of Dubensky etc.) of ICS and enzyme linked immunological spot assay method is disclosed.

When the carrier of using contained the nucleic acid of the ovalbumin of encoding, the analysis of the immunne response of inducing arbitrarily of the mode by ICS assay method or enzyme linked immunological spot assay method was used for from ovalbumin, OVA _257-264The standard peptide of (SIINFEKL (SEQ ID NO:l)) is wherein hatched with the peptide adding and with the splenocyte prepared product.

The nucleotide sequence of the people's mesothelin that uses in following construction is transferring the ENGINEERED LISTERIA AND METHODS OF USE THEREOF that submits on March 30th, 2006 of Cerus Corporation, in the United States Patent (USP) series number 11/395,197.The mesothelin peptide library of crossing over whole length of people's mesothelin is made of each 15-mer, overlapping 11 amino acid of each 15-mer and next 15-mer 153 kinds of peptides.

Identified Lm-hMeso38 (a kind of construct of the listeria monocytogenes that in following example, uses) in the table 1.Also referring to, the ENGINEERED LISTERIA AND METHODS OF USE THEREOF that submits on March 30th, 2006, United States Patent (USP) series number 11/395,197.Lm-mMeso is the construct identical with Lm-hMeso38, except total length people mesothelin sequence is replaced by total length mouse mesothelin sequence.Total length mouse sequence can be obtained by GenBank accession number NM_018857.

Table 3. listeria monocytogenes-hMeso38 (Lm-hMeso38)

The preparation of the listeria monocytogenes of coding OVA (" Lm-OVA ") is discussed in U.S. Patent Publication No. 2004/0197343 (United States Patent (USP) series number 10/773,618).

As described in (Overwijk etc. (1998) J.Exp.Med.188:277-286) by the N.P.Restifo preparation with the vaccinia virus source property carrier (VV-OVA) of the nucleic acid that comprises coding total length ovalbumin is provided.

The nucleic acid that contains coding total length people's mesothelin or total length mouse mesothelin (Ad-hMeso or Ad-mMeso) based on the carrier of adenovirus.Also use the contrast Ad carrier of the not encoding heterologous antigen that is called in addition " blank Ad carrier ".The Ad carrier of all contrasts and coding for antigens is all based on the adenoviral serotype 5 with E1 and E3 district disappearance, and all utilize " AdEasy " system that is obtained by Stratagem (San Diego, CA) to obtain and obtain according to the described method of supplier.With antigen clone at E1 locus place.The nucleic acid of encoding heterologous antigen is integrated in the shuttle vectors of the AdEasy that is connected with the operation of CMV promotor.

Refer to contain the tumour cell of inactivation of the nucleic acid of encoding murine rHuGM-CSF (GM-CSF), wherein tumor cell line is the CT-26 cell, expresses the clone of gp70.AH1 is the epi-position of gp70, a kind of immunodominant antigen of CT26 cell.As open (referring to, for example, Yoshimura etc. (2006) Cancer Res.66:1096-1104; Jain etc. (2003) Annals Surgical Oncol.10:810-820; Zhou etc. (2005) Cancer Res.65:1079-1088; Chang etc. (2000) Int.J.Cancer86:725-730; Borrello and Pardoll (2002) Cytokine Growth Factor Rev.13:185-193; Thomas etc. (1998) Human Gene Ther.9:835-843) prepares and use

(CELL GENESYS, INC.).

Prepare by the Lm alive that processes disappearance uvrAB gene and to kill but tool metabolic activity Lm (" KBMA-Lm "), use psoralene and ultraviolet ray, these expression together with the uvrC gene product form the required exonuclease of nucleotide excision reparation, this cause a small amount of genomic crosslinked (referring to, for example, United States Patent (USP) series number 10/773,618.The U.S. Patent Publication No. 2004/0197343.MODIFIED FREE-LIVING MICROBES of Dubensky etc., VACCINE COMPOSITIONS AND METHODS OF USE THEREOF; Brockstedt etc. (2005) Nature Med.11:853-860).

Embodiment 1

Use 1x10 ⁶The vaccinia virus of the coding OVA of pfu (" VV ") or use 5x10 ⁶The restructuring listeria monocytogenes (" Lm ") of the viral determinant of the disappearance actA of cfu and inlB and coding OVA is immunity C57BL/6 mouse (3 every group) in the time of the 0th day.Admit VV or the Lmat dosage of booster dose the 21st angel mouse, in each example, its with just exempt from immunizing dose and be equal to.Put to death mouse and at the 27th day results splenocyte.In order to be evaluated at the size of the afterwards vaccine-induced OVA specific C D8+T cell of single immunization, control mice was only admitted the single immunization of VV or Lm at the 20th day, then with its execution, and at the 27th day results splenocyte.In measuring, ISC uses OVA _257-264Peptide is measured OVA specific C D8+T cell response.

By just exempt from-OVA specific C D8+T cellullar immunologic response due to the strengthened scheme is presented among Figure 1A.As shown in FIG., when LM relatively just exempt from/when VV strengthens, utilize VV just exempt from/ first exempting from that Lm strengthens-strengthened scheme causes OVA specific C D8 ⁺The percentage of cell almost doubles.Just exempt from VV in addition ,/Lm adds persistent erection OVA specific C D8 ⁺The percentage homology Lm of cell just exempts from/and Lm strengthens high about 3 times; And than homology VV just exempt from/VV strengthens high about 9 times.These data are provided at heterology and just exempt from-strengthened scheme in direction-sense sign, obtain better result in the system of using Lm to strengthen.

Embodiment 2

Use the 3x10 of encoding human mesothelin ⁷The 5x10 of pfu adenovirus (" AV " or " gland-hMeso ") or use encoding human mesothelin (" Lm-hMeso38 ") ⁶Cfu Lm Δ actA/inlB injected 3 every group Balb/c mouse at the 0th day.At the 21st day, mouse, with in each example with just exempt from the dosage that immunizing dose is equal to and admit AV or the Lm-hMeso38 of booster dose.Put to death mouse and at the 27th day results splenocyte.In order to be evaluated at the size of the afterwards vaccine-induced OVA specific C D8+T cell of single immunization, control mice was only admitted AV and Lm-hMeso38 at the 20th day, then with its execution, and at the 27th day results splenocyte.

By just exempt from-vaccine-induced people's mesothelin specific C D4+ and CD8+ cellullar immunologic response due to the strengthened scheme be presented among Figure 1B.When with give Lm and just exempt from/one group of mouse of AV strengthened scheme is relatively the time, AV just exempts from/and Lm-hMeso38 strengthens high about 10 times of the percentile magnitude of the special spleen CD8+ cell of comparison people's mesothelin (" hMeso "), and this shows in the heterology strengthened scheme directed impact to the magnitude of the vaccine-induced cellular immunization of the antigen-specific of coding and obtain better result from the system of using Lm to strengthen.

Embodiment 3

At the beginning of admitting, Balb/c mouse (3 every group) exempts from dosage and booster dose to cause the immunne response to people's mesothelin.Use whole encoding human mesothelins shown in first the exempting from of carrier be illustrated among Fig. 2 with strengthened scheme.As from the result of ICS assay method as seen, AV just exempts from/ scheme that Lm strengthens produces and exempt from than Lm is first/ people's mesothelin (" hMeso ") specific C D8 that AV strengthens ⁺The percentage of cell is high 3 to 4 times.In addition, the result also shows in Fig. 2: and AV just exempts from/and any other heterologys that Lm strengthens being significantly higher than test just exempt from-strengthened scheme.

Embodiment 4

Tested different AV just exempt from dosage on the magnitude of vaccine-induced spleen hMeso specific C D4+ and CD8+T cell, just exempt from heterology AV/Lm adds the effect that the constant Lm of persistent erection strengthens.All vector encoded people mesothelins.HBSS is with comparing.First the exempting from of utilizing is illustrated among Fig. 3 A with strengthened scheme, and each experimental group is comprised of three C57BL/6 mouse.In these researchs, before ICS, will from the mouse of immunity, obtain splenocyte library, the mesothelin peptide pond stimulation 5 hours that the peptide fifteen amino acid (" 15x11 library ") by the skew of 4 amino acid lengths forms, this library, mesothelin peptide pond is corresponding to total length mesothelin albumen.Just exempting from-be illustrated among Fig. 3 A by the people's mesothelin specificity cellular immunity response due to the titration of AV in the strengthened scheme.As shown in the figure, under more substantial AV, obtain the remarkable increase that specific T-cells is replied, and CD8 ⁺T cell response is greater than CD4 ⁺T cell response.

Embodiment 5

Except just exempt from and strengthen between time be 38 days, and come the immunization control mice with Lm hMeso 38 but outside coming just to exempt from AV, in the Balb/c mouse, use to be similar to the immunization protocol (referring to Fig. 3 B) that in embodiment 4, uses.As shown in Fig. 3 B, specific T-cells is replied remarkable enhancing on the AV of wider scope amount; And again, CD8 ⁺T cell response is greater than CD4 ⁺T cell response.In the Balb/c mouse, using 5x10 ⁶After the Lm hMeso 38 of cfu strengthens, induce at AV and just exempt to be low to moderate 1x10 under the dosage ⁴The hMeso specific C D8+T cell of pfu basic horizontal.

Embodiment 6

In the presence of to the immunity that is pre-existing in of AV, test AV just exempts from/ validity that Lm strengthens.In for the first time research, test replying the AV immunity in the presence of the adenovirus specific immunity that is pre-existing in the scheme shown in Fig. 4 A.Before the AV immunity of using the encoding human mesothelin 28 days, cause the adenovirus specific immunity that is pre-existing in by " blank Ad carrier " the immunization mouse as shown in the figure of using various levels.5 Balb/C mouse/groups are used in this research.The 0th day to the blank Ad particle of injected in mice and subsequently at the 28th day, the AV (" Ad-hMeso ") of injection encoding human mesothelin.At the 35th day results splenocyte and be used in during ICS measures.Ad-blank and Ad-hMeso carrier are serotype 5.Stimulate splenocyte to measure the mesothelin specific immune response by end user's mesothelin peptide pond; Type of service I Hex3 epi-position is measured the AV specificity and is replied.Be presented at that the result shows among Fig. 4 A: in the presence of the Ad5 specific cellular immunity that is pre-existing in, when the Ad5 carrier that uses identical encoding human mesothelin during to mouse immune, only can induce low hMeso specificity to reply.

For the second time research test AV in the mouse that is pre-existing in the AV immunity just exempts from/the Lm strengthened scheme after hMeso specificity cellular immunity response (amount that shows the AV pfu that uses in the X-axis of Fig. 4 B).Except use AV just exempt from after 20 days, giving mouse with Lm-hMeso38 strengthens, and the alternative AV Specific T cell immunity of measuring is replied, test to measure outside the AV specificity neutralizing antibody by the plaque subtrahend, the scheme of use (being presented among Fig. 4 B) is similar to the scheme that test is just exempted from.When comparison diagram 4A, the result shows in Fig. 4 B: although to the existence of the neutralization immunity that is pre-existing in of AV serotype 5, the reinforcement of uniting Lm hMeso38 after AV just exempts from causes introducing the remarkable increase of hMeso specific cellular immunity.

Embodiment 7

Following use tumour cell just exempt from/ heterology that Lm strengthens just exempts from-strengthens.Scheme is used

(CELL GENESYS, INC.) just exempts from/the Lm-AH1-A5 reinforcement." GVAX " refers to contain the tumour cell of inactivation of the nucleic acid of coding rHuGM-CSF (GM-CSF).The listeria monocytogenes of using is Lm-Δ actA-OVA-AH1-A5.Lm-Δ actA-OVA-AH1-A5 is the restructuring listeria monocytogenes, and it comes attenuation by actA genetically deficient; And the antigen presentation box that contains single copy in the listeria genome; It is at tRNA ^ArgThe locus place is integrated, and wherein expression cassette contains insert AH1-A5 (Brockstedt etc. (2004) Proc.Natl.Acad.Sci.USA 101:13832-13837) in the ovalbumin inner frame.AH1-A5 is the AH1 L by the gp70 endogenous injections of antigens of CT26 gland cell system expression ^dThe T cytolipin of immunodominant epitopes's change, and be generally used for to tumour antigen (Slansky etc. (2000) Immunity 13:529-538 in the immunne response of gp70; Jain etc. (2003) Annals Surgical Oncol.10:810-820).

Just exempt from/strengthen for heterology, every group of 3 mouse admitted

(CELLGENESYS, INC.) (1x10 ⁶Individual cell) (s.c.) as just exempting from and Lm-OVA-AH1-A5 (5x10 ⁶Cfu) (i.v.) as strengthening.Just exempt from/strengthen vaccine for the homology with GVAX, mouse is admitted

(CELL GENESYS, INC.) just exempts from (1x10 ⁶Individual cell) (s.c) just exempt from and to strengthen being used for.Just exempt from/strengthen vaccine for the homology that only has Lm, give mouse Lm-OVA-AH1-A5 (5x10 ⁶Cfu) (i.v.) just exempt from and to strengthen being used for.Carry out independent homology and just exempt from/strengthen test, wherein only s.c, i.m. or i.v. use Lm.In all cases, in the time of t=0 days, carry out just exempting from; In the time of t=21 days, strengthen; Results splenocyte in the time of t=29 days.As shown in the figure, use (is only used separately single vaccine

(CELLGENESYS, INC.); Lm-OVA-AH1-A5 only) processes mouse.

Result's confirmation,

Lm-Δ actA-OVA-AH1-A5 after (CELL GENESYS, INC.) just exempts from strengthens the AH1 specific immune response that causes than just exempting from by any homology of using of any approach/booster immunization scheme larger.

Embodiment 8

Following carry out AV just exempt from/Lm strengthens just exempting from dendritic cell (" DC ")/ comparison that Lm strengthens.Use every group of 5 Balb/c mouse.With 2x10 ⁶The amount of individual dendritic cell (i.v.) is used dendritic cell.To contain the adenovirus source property carrier of nucleic acid of encoding human mesothelin with 1x10 ⁸The dosage of cfu is used (i.m.).With Lm-hMeso38 with 5x10 ⁶The amount of cfu is used (i.v.).With the DC of peptide pulse with 2x10 ⁶The dosage of DC is used (i.v.).DC and Lm just exempt to be separated by 8 days with booster immunization, and AV and Lm just exempt to be separated by 14 days with booster immunization.In all mouse, a couple of days is measured spleen mesothelin 131-139 specific C D8+T cell afterwards in the booster immunizationization with Lm hMeso 38.Contrast is illustrated among Fig. 6 A and Fig. 6 B.

Be prepared as follows the DC of peptide pulse.Use hMeso _{131_139}(SGPQACTRF) come pulsed D C.Use high GM-CSF concentration (20ng/mL mouse GM-CSF) (R﹠amp; D Systems, Minneapolis, MN) from the whole marrow of Balb/c mouse, prepare DC.After carrying out initial inoculation and GM-CSF enrichment the 8th day, gather in the crops non-adhesion T cell and verify that through phenotype it is marrow dendritic cell (CD11c ^Hi).Use lipopolysaccharides (LPS) to process DC (24h) and use 1.0 micromole's peptide (hMeso _{131_139}) (0.001mM) pulse 1 hour.Before 1x106DC being injected to acceptor Balb/c mouse, with the DC washed twice of peptide carrying.

The result who measures at the ICS shown in Fig. 6 A confirms: have DC-hMeso 131-139 just exempt from/ heterology that Lm-hMeso38 strengthens is just exempted from/is strengthened and allos sexual gland-hMeso just exempts from/Lm-hMeso38 strengthens each self-induction mesothelin specificity cellular immunity response, wherein to exempt from the beginning of two kinds/level of the immunne response of strengthened scheme is basic identical.

Be presented at enzyme linked immunological spot assay method among Fig. 6 B The resultConfirm: have DC-hMeso131-139 just exempt from/ heterology that Lm-hMeso38 strengthens is just exempted from/is strengthened and allos sexual gland-hMeso just exempts from/Lm-hMeso38 strengthens each self-induction mesothelin specificity cellular immunity response, wherein for exempt from the beginning of two kinds/the immunne response level of strengthened scheme is basic identical.

Embodiment 9

Carry out following DC just exempt from after relatively as Lm, the AV and the VV that strengthen.At the 0th day, with Balb/c mouse (five every group) intravenous inoculation 1x10 ⁶Individual bone marrow derived dendritic cell (just exempting from).Prepare as described in Example 8 DC.At the 21st day, provide reinforcement to mouse, it is one of following wherein strengthening: Lm-hMeso38 (5x10 ⁶Cfu, i.v.); Ad-hMeso (1x10 ⁷Pfu, i.m.) or vaccinia virus-hMeso (" VV-hMeso ") (1x10 ⁶Pfu, i.p.).HBSS is received in independent mouse winding, rather than strengthens vaccine.After booster shot, measured Meso by cell within a cell factor dyeing (ICS) in 5 days _131-139Specific C D8 ⁺T cell response.

Be presented at that the result shows among Fig. 6 C: although the AV-hMeso disappearance provides the hMeso cell-specific is replied more remarkable reinforcement, obtain about 5 times of higher results with Lm as stiffeners.

Embodiment 10

Relatively use Lm just exempt from as the heterology of strengthening-strengthened scheme in as AV and the naked DNA of just exempting from.In addition, the mouse mesothelin is substituted people's mesothelin whether can just exempt from heterology to measure-strengthened scheme destroys tolerance.Under study for action, use the Balb/c mouse.Use heterology DNA just to exempt from/Lm strengthens and heterology Ad just exempts from/and Lm strengthens vaccine, assesses the mesothelin specific immune response with whole mesothelin peptide library.DNA just exempts from/and it is that DNA-mMeso (0.1mg) (i.m.) just exempts from/Lm-mMeso38 (5x10 that Lm strengthens ⁶Cfu) (i.v.) reinforcement (Fig. 7 A).Ad just exempts from/and it is Ad-mMeso (1x10 that Lm strengthens ⁸Pfu) (i.m.) just exempt from/Lm-mMeso38 (5x10 ⁶Cfu) (i.v.) reinforcement (Fig. 7 B).Be prepared as follows the naked DNA carrier, DNA-mMeso.The nucleic acid of coding total length mouse mesothelin is inserted in the pCDNA3 (a kind of carrier for expression of eukaryon) (Invitrogen Corp., Carlsbad, CA).

Used first exempting from the 0th day; Used reinforcement at the 13rd day; At the 18th day results splenocyte.Use is processed the splenocyte of results from the peptide of mouse mesothelin (" mMesothelin ") peptide library, and wherein the peptide adding with uniqueness respectively contains in the hole of splenocyte, and wherein assesses immunne response with enzyme linked immunological spot assay method.Be used to from the hole of the sufficient amount of the splenocyte of immunization mouse, so that can use the unique peptide (fifteen amino acid is long, is offset 4 amino acid) of the total length that comprises mouse mesothelin albumen, so that t cell responses mouse mesothelin peptide can be identified.

The result shows in Fig. 7 A: and when each peptide in the mMesothelin peptide library was assessed immunne response separately, DNA-mMeso just exempted from/and Lm-mMeso38 strengthens causing relatively less immunne response.Otherwise the result in Fig. 7 B shows: and Ad-mMeso just exempts from/and Lm-mMeso38 strengthens inducing to the relatively high immunne response from the mouse mesothelin peptide numbering 278,279 and 280 of mouse mesothelin peptide library.Find relatively low immunne response, wherein use other peptides from mouse mesothelin peptide library.Fig. 7 C is the photo in hole, wherein splenocyte is exposed to mesothelin peptide numbering 278,279 or 280, and wherein photo illustrates spot from enzyme linked immunological spot assay method.Therefore, being presented among Fig. 7 B and Fig. 7 C result confirms: and AV just exempts from/and the Lm strengthened scheme causes the specificity cellular immunity response to mMesothelin.And the result also is illustrated in the first mouse of exempting from the booster immunization scheme that gives to be comprised of two carrier A V of encoding murine mesothelin and Lm carrier the destruction to the tolerance of mouse mesothelin endogenous antigen.

Embodiment 11

Assessment is as the effect of just exempting from the KB MA Lm of agent and/or stiffeners.Be used as the KBMA-Lm that just exempts from the 0th angel and use C57BL/6 mouse (3 every group).Gave one of following reinforcement at the 14th day: " work " Lm; KBMA-Lm; Or VV.Also used KBMA-Lm at the 14th day and the contrast of the Lm that lives.Put to death mouse at the 19th day, then gather in the crops splenocyte.

The listeria construction has hly promotor and the ovalbumin that is operatively connected to the BaPA secretion sequence, its The place has genome conformity at the tRNAArg locus.

The number of the KBMA listeria monocytogenes Δ actA Δ uvrAB-OVA (KBMA Lm) of injection is 3x10 ⁸Individual bacterium particle.KBMA Lm is through psoralene and UV optical processing.The amount of " work " listeria monocytogenes of using (Lm-Δ actA Δ uvrAB-OVA) is 1x10 ⁶Cfu.The amount of the vaccinia virus source property carrier (VV-OVA) of using is 1x10 ⁶Pfu.

By using by ovalbumin (OVA _257-264) mode of the peptide-pulse of the standard octapeptide of deriving and cell inner dyeing (ICS) assay method by IFN-γ measures the ovalbumin specific immune response and measure immunne response.

As shown in the result being presented at Fig. 8, KBMA-Lm is the active agent of just exempting from.KBMA-Lm just exempts from/lives Lm and strengthens causing higher levels of immunne response, wherein use heterology KBMA-Lm just exempt from/VV strengthens vaccine and lower level immunne response occurs.

Embodiment 12

With in every group of 15 Balb/c mouse muscles the inoculation 1e8pfu blank adenovirus (namely, do not express the adenovirus of AH1, AH1/A5 or mMesothelin) or express the adenovirus of AH1/A5 or mouse mesothelin (mMesothelin), after 21 days, with the Lm of the 5e6cfu of encoding heterologous antigen or the Lm of expression AH1/A5 or mMesothelin do not come intravenously to strengthen mouse.AH1/A5 (referring to, for example, Brockstedt etc. (2004) Proc.Natl.Acad.Sci.USA 101:13832-13837) has been described.After 5 days, obtain the spleen of every group of 5 mouse with assessment AH1/A5 (Fig. 9 A) and mMesothelin (Fig. 9 B) immunne response.In a week after booster shot, use 4e5 CT26 cell intravenously to excite all the other mouse.The survival rate (Fig. 9 C) of monitoring mouse.In Fig. 9 A-9C, " Ad " represents blank adenovirus; " Lm " represents the not listeria monocytogenes of encoding heterologous antigen; The adenovirus of AH1/A5 is expressed in " Ad-AH1/A5 " expression; The adenovirus of mouse mesothelin is expressed in " Ad-mMeso " expression; The listeria monocytogenes of " Lm-AH1/A5 " presentation code AH1/A5; And the listeria monocytogenes of " Lm-mMeso " presentation code mouse mesothelin.

As shown in Fig. 9 A, according to the ICS assay method, use Ad-AH1/A5 just to exempt from a large amount of antigen-specific CD8+T cellullar immunologic responses of first the exempting from strengthened with Lm-AH1/A5/strengthen causing.As shown, AH1 peptide and AH1/A5 peptide are derived by gp70 (a kind of endogenous protein of CT26 tumour cell).As shown, replenishing splenocyte with AH1 peptide or AH1/A5 peptide hatches.Gp70 is the endogenous protein of CT26.When splenocyte is hatched with the AH1/A5 peptide of interpolation, has larger CD8+T cellullar immunologic response; And then has less CD8+T cellullar immunologic response when hatching with the AH1 peptide that adds.

The specificity of following confirmation immunne response.Use blank adenovirus and the detectable CD8+T cellullar immunologic response of first the exempting from of the Lm of encoding heterologous Ag/strengthen can not causing not.And, first the exempting from of the carrier of use expression mouse mesothelium/strengthen can not causing detectable AH-or AH1/A5 specific C D8+T cellullar immunologic response, this reconfirms the specificity (Fig. 9 A) of immunne response.

Be presented at data acknowledgement among Fig. 9 B to another heterogenetic antigen, i.e. first the exempting from of the immunne response of mesothelin/add strong stimulation; And immunne response is to this antigen tool specificity.Specificity is shown, namely blank Ad just exempt from/Lm (not encoding heterologous Ag) strengthens unsuccessfully; Perhaps Ad AH1/A5 just exempt from/Lm AH1/A5 adds strong stimulation mMesothelin specific C D8+T cellullar immunologic response.

Just exempt from the data acknowledgement shown in Fig. 9 C: Ad-AH1/A5/Lm-AH1/A5 strengthen to increase the survival rate that excites with the CT26 tumour cell.Accompanying drawing also confirms: and Ad-mMeso just exempts from/and Lm-mMeso increases the survival rate that the CT26 tumour cell excites.As processing relatively (Fig. 9 C) with using HBSS, use blank carrier (blank Ad; The Lm of expressing heterologous Ag not) processes and just exempt to strengthen not changing survival rate.

Embodiment 13

Research reagent, CRS-207 is comprised of the bacterial strain of the work-attenuation of bacterium listeria monocytogenes (LM) in the cell of codes for tumor antigen mesothelin (Lm Δ actA/ Δ inlB/hMeso).The purpose of using CRS-207 is to reply by the inducing antitumor of inducing of cell-mediated immunity (CMI), and this cell-mediated immunity (CMI) is for expressing such as the mesothelin on the cell surface of malignant mesothe, nonsmall-cell lung cancer (NSCLC) and carcinoma of the pancreas and ovarian cancer.Obtain CRS-207 by disappearance from whole encoding sequences of the genomic two-strain of wild-type Lm-determinant gene.Two-strain-determinant gene, the product of inlB and actA are conducive to respectively nonphagocytic Lm invasion and cell to cellular invasion.As passing through the viral assessment in the mouse, the merging disappearance of these two kinds of encoding sequences causes attenuation to surpass 1,000 times.Yet, the picked-up of CRS-207 be retained and cause local inflammation to reply and activate and raise immune effector cell to liver, for example natural killer (NK) cell and T cell by the scavenger cell in liver and spleen and other phagocytic cells.After by the picked-up of phagocytic cell (comprising DC and scavenger cell) to CRS-207, mesothelin is expressed and is released in the cytosol interval, and subsequently its processing is presented approach by endogenous MHC I class, this causes the activation of the cell-mediated immunity of anti-mesothelin (CMI).Other mechanism that activate mesothelin specific C MI can be included in to be passed through after CRS-207 infection and the apoptosis from the APC picked-up of scavenger cell and/or other cell types and the antigen-presenting that intersects.

CRS-207 is formulated as two kinds of concentration: high dosage and low dosage.Select two kinds of concentration so that the dosage range that preparation is expected in clinical study.Study reagent referring to table 1a and 1b to be used for preparation CRS-207.

Table 1a CRS-207 high dosage formulation

Table 1b CRS-207 low dosage formulation

With CRS-207 refrigerated storage under-75 ℃ or lower temperature.It is comprised of the Lm of the attenuation in the Dulbecco phosphate-buffered saline (DPBS) that is suspended in 1.5mL and the 9%v/v glycerine.Each high dosage bottle has 5x10 ¹⁰The concentration of cfu/mL and each low dosage bottle have the concentration of 1x108cfu/mL.

CRS-207 is delivered among people experimenter's (〉=18 one full year of life) of the terminal cancer with ovary or pancreas, nonsmall-cell lung cancer or malignant mesothe, these experimenter's Application standard therapeutics failures or its are not the candidate targets of standard care method.The experimenter who includes this research in meets all following choice criteria:

Through standard care failure or be not on the histology of candidate target of standard care or the malignant mesothe that records on the cytology; The gland cancer of pancreas; Perhaps express nonsmall-cell lung cancer (NSCLC) or the ovarian cancer of mesothelin.

ECOG performance score 0-1 or function of human body state scale (Karnofsky Performance Status) (KPS) 80% to 100%.

The time length that research is longer than in expection life expectancy.

In research phase of all experimenters and after using CRS 207 28 days during in, female subjects that may be conceived and all male subject must agree to use the highly effectively medically acceptable method (oral hormone contraception, condom and spermicide or hormone graft) of contraception.Regardless of additive method, must comprise the barrier approach of contraception.

The experimenter provides Informed Consent Form and is ready and can observes all research operations.

Sufficient organ dysfunction limits as follows:

Hematology

Thrombocyte 〉=100x10 ⁹/ L

Oxyphorase 〉=9.0g/dL

Total WBC counting 〉=3.5x10 ⁹/ L

ANC≥1.5x109/L

Total lymphoblast counting 〉=0.8x10 ⁹/ L

PT/INR and PTT≤ULN of 1.3x clinical labororatory

Liver

Bilirubin≤the ULN of 1.5x clinical labororatory

AST and ALT≤ULN of 2.5x clinical labororatory (for the patient with carcinoma of the pancreas, the ULN of≤3.5x clinical labororatory is acceptable)

GGT≤the ULN of 5x clinical labororatory

Alkaline phosphatase≤the ULN of 2.5x clinical labororatory

Kidney

Serum creatinine≤the ULN of 1.5x clinical labororatory

In addition, the experimenter can not have following exclusion standard:

The known central nervous system that is transferred to.

Li Siteshi medical history or use are based on the inoculation medical history of the vaccine of listeria.

Known to penicillin anaphylaxis.

Clinical remarkable heart trouble (for example congestive heart failure of uncontrolled stenocardia, myocardial infarction, New York heart association III or IV in 3 months).

Individuality with valvular heart disease, it needs Antibiotic prophylaxis to be used for the prevention endocarditis consistent with AHA guide Circulation such as (, 1999) Dajani.

As measure O2 saturation ratio＜92% in air at room temperature by pulse oximeter.

Do not have selected study condition based on medical history and physical examination impaired lung function or doubtful impaired research experimenter, unless the desired value of record VC, DLCO and FEV1〉60%.

Need active, the clinical significance autoimmune disorder of whole body therapeutic or the medical history of autoimmune disease.

Clinical test results corresponding to 3 grades or the 4 grades clinical metabolism/laboratory abnormalities of CTCAE.

Liver cirrhosis or clinical associated Ascites or the individuality (for example, the tumor quality by hepatic portal reduces) with a large amount of risks of obstructive jaundice.

Manually (prosthese) joint or other artificial graft or the devices that can not remove easily.If do not have historical with not relevant with the graft clinical remarkable side effect of graft infection to occur, can allow tooth transplantation thing and breast transplant.

The known disorder or take the antithrombotics medicine of condensing.

The patient that need to inculcate than twice more frequent routine per month.

Blood transfusion in 14 days of selected research is except unprovoked oligoleukocythemia and radiation.

Any immune deficiency disorder or immunocompromised host situation (for example, are used immunosuppressor; In 28 days before using CRS 207 or in chemotherapeutics or the radiation-therapy of using CRS 207 plans in rear 28 days).

Plan in 28 days before using CRS 207 or in using during the research after the CRS 207 uses the system activity steroid more than 2 days.In using 14 days of CRS 207, use any system class sterol

The female subjects of pregnancy or lactation.The conceived female subjects of possibility must have negative β-hCG and test (serum or urine).

The illegal medicine (for example, opiates, Cocaine, amphetamines, halluoinogen etc.) of alcohol dependence history or use possibility the Study of Interference rules or requirement.

Use CRS 207 in the internal jugular vein at 2 hours and treat the experimenter.As shown in following table, increase the dosage of each successive doses group.

When the dosage of research increases part, use CRS-207 with 21 days intervals between dosage.

The experimenter is divided into those experimenters in the first time of using CRS 207 of survival rate 〉=15 month after the consumption, and has those experimenters of survival rate＜15 month after the dosage in the first time of using CRS 207.Following table is presented at the demographic statistics in these two groups.

Improve for survival rate among the experimenter of adjuvant therapy at CRS 207.

Admitted before in those were tested in the II phase

Among the experimenter of (CELL GENESYS, INC.), improve survival rate by sending subsequently CRS 207:

One of ordinary skill in the art will readily recognize that the present invention is easy to adapt to finishes purpose and the advantage that theme and acquisition are mentioned, and intrinsic those of this paper.The example that this paper provides is representative preferred embodiment, and it is exemplary, and is not intended to limit the scope of the invention.

Although fully described in detail and exemplified for those skilled in the art and how to have carried out and use it, not run counter under the spirit and scope of the present invention, various alternative forms, change and improvement should be obvious.The example that this paper provides is representative preferred embodiment, and it is exemplary, and is not intended to limit the scope of the invention.Those skilled in the art can expect modification and other purposes wherein.These changes are encompassed in the spirit of the present invention, and by the circumscription of claim.

It will be apparent to those skilled in the art that and do not running counter under the spirit and scope of the present invention and can carry out different replacements and change to the present invention disclosed herein.

The level of all patents of in specification sheets, mentioning and publication indication the technical field of the invention those of ordinary skill.All patents and publication all are incorporated herein by reference, and it quotes degree as pointing out that clearly and individually each independent publication incorporates into way of reference.

The present invention of this paper schematic description can lack any or Various Components, non-concrete disclosed one or more restrictions of this paper under carry out.Therefore, for example under each example of this paper, term " comprises ", " substantially by ... form " and " by ... form " in any can replace in twos each other or between the term.The term that has adopted and expression are as the term of describing; and be nonrestrictive; and use these terms and express be not be intended to get rid of shown in and any equivalents of described feature or its part; but recognize that the various changes in the present invention's scope required for protection all are possible.Therefore, be to be understood that, although by preferred embodiment and specifically openly the present invention of optional feature, those skilled in the art can obtain change and the version of concept disclosed herein, and think that these changes and variation are all within the scope of the invention that is defined by the following claims.

Other embodiments are illustrated in the claims.

Claims (11)

1. one kind is used for suffering from cancered mammiferous method of assisting a ruler in governing a country treatment, and described method comprises:

Treat to remove or kill the cancer cell of expressing cancer antigen as assisting a ruler in governing a country at the composition to described administration significant quantity before or after the main therapy of described administration, described composition comprise attenuation, the metabolic activity listeria, described attenuation, metabolic activity listeria encode effable, the immunocompetence part of described cancer antigen.

2. method according to claim 1, wherein applying said compositions after described main therapy.

According to claim 1 and 2 in each described method, wherein said main therapy comprises from described Mammals the operation of removing described cancer cell; Kill the radiation-therapy of the described cancer cell in the described Mammals; Perhaps described operation and described radiation-therapy the two.

4. each described method according to claim 1-3, wherein said step of applying comprise with multiple doses use described attenuation, the metabolic activity listeria, described attenuation, metabolic activity listeria encode effable, the immunocompetence part of described cancer antigen.

5. each described method according to claim 1-4, wherein said attenuation, the metabolic activity listeria has the sudden change that makes the ActA inactivation.

6. each described method according to claim 1-5, wherein said attenuation, the metabolic activity listeria has the sudden change that makes the InlB inactivation.

7. each described method according to claim 1-6, wherein said attenuation, the metabolic activity listeria is Δ actA Δ inlB.

8. each described method according to claim 1-7, wherein said listeria is killed but tool metabolic activity (" KBMA ").

9. each described method according to claim 1-8, wherein said cancer antigen are whole or a part of mesothelins.

10. each described method according to claim 1-9, wherein before described step of applying, expressed in advance the cell from the human cell line of whole or a part of described cancer antigen to described administration, the reorganized modification of wherein said cell is with generation and secretion rHuGM-CSF, and wherein said cell is modified in case cell fission.

11. each described method according to claim 1-10, wherein said cancer is selected from: carcinoma of the pancreas, nonsmall-cell lung cancer, ovarian cancer and mesothelioma.

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